Issued September 7, 1912; 



a7. 
Co, 



U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— BULLETIN 151. 

A. D. MELVIN, Chibp op Bureau. 



A STUDY OF THE GASES OF 
EMMENTAL CHEESE. 



BY 



WILLIAM MANSFIELD CLARK, Ph. D., - 

Chemist, Dairy Division. 




WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 

1912. 



'sm 




Glass S F^l 
Book C ( 



-/ hM 



Issued September 7, 1912. 

U. S. DEPARTMENT OF AGRICULTURE, 

BUREAU OF ANIMAL INDUSTRY.— Bulletin 151. 

A. D. MELVIN, Chief of Bureau. 



A STUDY OF THE GASES OF 
EMMENTAL CHEESE. 



BY 



WILLIAM MANSFIELD CLARK, Ph. D., 

Chemist, Dairy Division. 




WASHINGTON: 
GOVERNMENT PRINTING OFFICE. 

1912. 






cfb 



'J 



THE BUREAU OF ANIMAL INDUSTRY. 



Chief: A. D. Melvin. 

Assistant Chief: A. M. Farrington. 

Chief Cleric: Charles C. Carroll. 

Animal Husbandry Division: George M. Kommel, chief. 

Biochemic Division: M. Dorset, chief. 

Dairy Division: B. H. Rawl, chief. 

Field Inspection Division: R. A. Ramsay, chief. 

Meat Inspection Division: Rice P. Steddom, chief. 

Pathological Division: John R. Mohler, chief. 

Quarantine Division: Richard W. Hickman, chief. 

Zoological Division: B. H. Ransom, chief. 

Experiment Station: E. C. Schroeder, superintendent. 

Editor: James M. Pickens. 

DAIRY DIVISION. 

B. H. Rawl, Chief. 

Helmer Rabild, in charge of Dairy Farming Investigations. 
S. C. Thompson, in charge of Dairy Manufacturing Investigations. 
L. A. Rogers, in charge of Research Laboratories. 
Ernest Kelly, in charge of Market Milk Investigations. 
Robert McAdam, in charge of Renovated Butter Inspection. 
2 



ADDITIONAL COPIES of this publication 
I*- may be procured from the Superintend- 
ent of Documents, Government Printing 
Office, Washington, D. C, at 5 cents per copy 



OC^ 8 rcn? 



LETTER OF TRANSMITTAL. 



U. S. Department of Agriculture, 

Bureau of Animal Industry, 

Washington, D. C, April 23, 1912. 

Sir: I have the honor to transmit, and to recommend for publi- 
cation in the bulletin series of the bureau, the accompanying manu- 
script entitled "A Study of the Gases of Emmental Cheese," by Dr. 
William Mansfield Clark, chemist in the Dairy Division. 

The so-called "eyes" in Swiss cheese are, as is well known, its most 
prominent characteristic, and its commercial value is largely depend- 
ent upon the proper size and spacing of these eyes. Furthermore, 
much depreciation in the value of this popular variety of cheese, in 
both the domestic and foreign kinds, is known to exist because of 
defects in eye formation. The experimental work herein described 
concerns the chemical contents of these eyes, and although consider- 
able work has been done in Europe with the object of discovering the 
cause of eye formation, there has hitherto been no investigation made 
of the gases which are immediately concerned in the process. Dr. 
Clark's studies are therefore calculated to be of value to the scientific 
as well as the practical side of the industry. 

Kespectfully, 

A. D. Melvin, 

Chief of Bureau. 
Hon. James Wilson, 

Secretary of Agriculture. 

3 



CONTENTS. 



Page. 

Introduction 7 

Description of apparatus and methods of collecting the gases 9 

Method 1 9 

Method II 10 

Method of analysis 11 

Discussion of the analyses 12 

Absorption of oxygen 18 

The permeability of cheese to gases 20 

Nitrogen dissolved in curd 24 

Does nitrogen originate in situ? 25 

Relation between carbon dioxid and volatile acids 26 

Summary 31 

References to literature 32 

5 



ILLUSTRATIONS. 



Page. 

Fig. 1. Apparatus for collecting gas from the eyes of Swiss or Emmental cheese . . 8 

2. Apparatus for pumping gas from cheese 10 

3. Apparatus for studying the absorption of oxygen by cheese 14 

4. Device for ascertaining permeability of cheese to gases 20 

5. Apparatus for determining amount of nitrogen in curd 24 

6 



A STUDY OF THE GASES OF EMMENTAL CHEESE. 



INTRODUCTION. 

The " eyes" of Swiss or Emmental cheese are its most striking 
characteristic. Their formation is a fascinating subject to the bio- 
logical chemist, because of a supposed localization of reactions gen- 
erating considerable quantities of gas, and because of the produc- 
tign of a plasticity among the colloids of the cheese, which makes 
possible the peculiar mold of the cavities. 

To the cheese maker the formation of the "eyes" is a matter of 
great importance, since their size and proper spacing determine in 
large measure the commercial value of the cheese. In certain dis- 
tricts of Wisconsin visited by the writer the dealers rely almost 
entirely upon these features, and, shortly after the eyes have reached 
their proper development, relieve the maker of further care. The 
American makers of Swiss cheese are, therefore, unable to attend 
to their cheeses in that mellow old age upon which so much of the 
fine flavor of a true Emmental cheese depends. However much this 
quick marketing is to be deprecated, the fact remains that it raises 
the relative importance of the eye formation and adds significance 
to whatever knowledge can be gained concerning the process. 

Some years ago Bachler, 1 ° cited by Jensen, 11 estimated that 25 
per cent of the cheeses made in Switzerland were considerably reduced 
in value because of imperfect eye formation. How far this enormous 
loss has been lessened in recent years as a result of scientifically con- 
trolled manufacture can not be said, but in this country, where large 
numbers of Swiss are still using the antiquated methods of their fore- 
fathers, Bachler's estimate is probably not too high. The wide dif- 
ference in market price between domestic and imported Swiss cheese 
bears out this statement. 

Considerable work has been done in Europe in the effort to uncover 
the cause of eye formation, and, through the labors particularly of 

a The reference figures relate to the list of references to literature at end of bulletin. 

7 



8 



STUDY OF GASES OF EMMENTAL CHEESE. 



Von Freudenreich and Jensen, a well-founded theory has been proposed 
which will be discussed later. No one, however, has made a study of 
the gases which are themselves the immediate cause of the eye forma- 
tion, and it was with the hope that such a study might furnish valu- 
able data that the research herein described was undertaken. If 
nothing more is demonstrated than the composition of the gas in 




#**>■•: 



Fig. 1.— Apparatus for collecting gas from the eyes of Swiss or Emmental cheese. 

the eyes, this alone justifies the work, for the extensive researches on 
the eye formation in Emmental cheese have led to but one conclu- 
sion that can be called positive, and that is that a final explanation 
will be reached only when every phase of the subject has been sub- 
mitted to exact quantitative study. 



APPAEATUS AND METHODS. 9 

DESCRIPTION OF APPARATUS AND METHODS OF COLLECTING THE 

GASES. 

The collection of the gas in the eyes by cutting the cheese under a 
bell jar filled with water, as was done with Edam cheese by Boekhout 
and Ott de Vries, 2 is a simple and valuable method, but one which 
is hardly to be called accurate, owing to the high solubility of certain 
gases in water. In place of such a method an apparatus was devised 
for collecting the gas over mercury. This is shown in figure 1, the 
procedure being as follows: 

METHOD I. 

The glass cylinder A is forced a short distance into the body of 
the cheese until it is firmly held. It is then clamped in position. 
Around the outside the cheese is cut away sufficiently to leave a 
channel into which mercury moistened with mercuric chlorid solution 
is poured. This forms a seal preventing entrance of air. The head 
of the shaft B is now resting on the surface of the cheese. Through 
its capillary mercury is run into the cylinder, displacing the air until 
it finally runs out of the side arm D and up through the annular 
space between the shaft and the shoulder of the cylinder. The short 
length of thick rubber tubing at E is then very tightly bound with a 
rubber band, leaving mercury in the small cup above, and thus 
effectually closing this opening against the entrance of air. When 
the cylinder and side arm are thus completely filled with mercury, a 
receptacle filled with mercury is brought over the end of the side 
arm (in a mercury trough, of course) and serves to retain the col- 
lected gas until the time of the analysis. After these preparations 
the shaft is pushed down into the cheese. When it punctures an 
eye this can readily be felt. Since the head of the shaft is larger 
than the shank, there is left an annular space for the escape of the 
gas. This gas is displaced from the eye partially by the mercury of 
the cylinder, which finds its way to the lower level, but more largely 
by the mercury which runs in through the capillary in the shaft. The 
exit of this is prevented from becoming clogged with cheese by care- 
fully blowing it out just behind the head, as shown in the diagram. 
When the gas is displaced from the eye it is displaced from the 
cylinder into the receiver by continuing to run in mercury through 
the shaft from the reservoir C. Between this reservoir and the shaft 
is placed a bulb which prevents the mercury from sweeping in bub- 
bles of air. 

In the samples of gas collected with this apparatus seldom was more 
than a trace of oxygen found. This in itself shows that the gas was 
obtained without contamination by air. 
42208°— Bull. 151—12 2 



10 



STUDY OF GASES OF EMMENTAL CHEESE. 



METHOD II. 

For the collection of gas from "pinholes" the foregoing apparatus 

was of little use except in one instance to be mentioned later. To 

collect the gas from this form of hole, as well as the gas in the body 

of the cheese, the apparatus shown in figure 2 was used, as follows : 

Samples of cheese taken with a trier were introduced into the 

glass cylinder A. The rubber 
stopper at B, attached to the 
mercury vacuum pump with or 
without the intermediate connec- 
tion C, was forced in securely and 
protected from leakage by the 
mercury seal. Upon raising the 
leveling bulb D the cheese was 
flooded with mercury and the sur- 
rounding air was forced over into 
the pump until the mercury stood 
at the stopcock E. To prevent 
bubbles of air being trapped under 
the cheese the lower ends of the 
plugs were sharply beveled. Bub- 
bles of air of course adhered to the 
rough surface of the cheese and its 
smaller exposed cavities. This 
error is inherent in the method, 
but was reduced by suddenly drop- 
ping the leveling bulb with the stop- 
cock E closed, and then driving the 
air, which had expanded into the 
vacuum, past the open stopcock. 
The glass tube with its trap 
which connects A with the level- 
ing bulb was made sufficiently 
long so that D might be lowered 
the barometric distance below A, 
and thus leave the cheese exposed 
to a fairly high vacuum even be- 
fore the pumping commenced. After exhausting the pump up to 
E this cock was opened, and the gas pumped from the cheese and 
delivered into a receiver. 

The mercury pump used in this as in other operations to be de- 
scribed later was AntropofFs modification of the Topler. A full 
description of the pump and its appurtenances will appear in the 
account of another investigation. 




Fig. 2.— Apparatus for pumping gas from cheese. 



METHOD OF ANALYSIS. 



11 



METHOD OF ANALYSIS. 

The gas was analyzed with, a special set of burettes and pipettes 
designed for the analysis of small quantities of gas produced by 
bacteria. A few of the first analyses were made with a burette spe- 
cially designed for volumes as low as 0.5 c. c. In all the analyses 
the confining liquid was mercury, and use was made of a device for 
extremely accurate separation of gas from absorbent. 

Thirty-three per cent potassium hydroxid solution, in quantities 
appropriate for the volume of gas analyzed, served as absorbent for 
carbon dioxid. Hydrogen sulphid, after prehminary qualitative 
tests, was assumed to be absent, although it is of course possible 
that, if present originally in the gas, it may have been taken up by 
the mercury. That any of this gas occurs in the eyes is, however, 
very improbable, for its odor was never detected. For hydrogen 
sulphid and mercaptans the nose is many times more sensitive than 
is the spectroscope for sodium, 6 and unless the other and milder 
odors of Swiss cheese exercise a surprisingly intense hindrance to the 
detection of hydrogen sulphid and mercaptans we may justly say 
that these vapors were absent. With Nessler's reagent very slight 
traces of ammonia were detected. For oxygen alkaline pyrogallol 
or long-continued contact with phosphorus was used. Combustible 
gases were estimated in several ways. Explosion with oxygen, in 
the presence of electrolytic gas when necessary, was used in several 
instances. For one case combustion with a platinum sponge was 
tried. For the small percentages of combustible gases found the 
method of Dennis and Hopkins 5 was found to be the most satis- 
factory. This consists, essentially, in leading the gas slowly into a 
measured volume of oxygen and there burning it slowly and quietly 
with a platinum wire heated by an electric current. 

Table 1. — Analyses of gas collected by puncturing apparatus from eyes of Swiss (Em~ 

mental) cheese — Method I. 



Desig- 
nation 

of 
cheese. 


Total 
vol- 
ume of 
gas col- 
lected. 


Contraction- 


Composition. 




Due to 
absorp- 
tion 
with 
KOH. 


Due to 
absorp- 
tion 
for 2 . 


Due 
to 
com- 
bus- 
tion. 


co 2 . 


o 2 . 


Hydro- 
car- 
bons. 


H 2 . 


N 2 . 


Description of cheese. 




C.c. 

0.96 

2.73 

1.66 

4.77 

1.25 

/ 3.44 

\ 4.02 

15.24 

f 7.56 

1 4.99 

/ 14. 47 

\ 9.99 

5.42 

4.96 


C.c. 
0.55 
2.29 
1.11 
2.44 
1.00 
2.23 
2.52 

13.77 
6.14 
4.04 

12.91 
8.95 
3.04 
2.35 


C.c. 


C.c. 


Per 

cent. 
57.3 
83.9 
66.9 
51.2 
80.0 
64.8 
62.7 
90.4 
81.2 
80.9 
89.2 
89.5 
56.0 
47.4 


Per 
cent. 


Per 
cent. 


Per 
cent. 


Per 
cent. 


Imported, eyes normal. 
Do. 


b 


0.00 
.01 
.02 
.00 
.00 

"".'66' 

.02 
.03 
.03 
.01 
.02 
.01 


'o.'io' 

.02 
.00 
.60 
.45 
.76 
.43 
.30 
.00 
.09 
.06 
3.63 


0.0 

Trace. 

Trace. 
0.0 
0.0 

""6.' 6' 

Trace. 
0.6 
0.2 

Trace. 

Trace. 

Trace. 








c 
d 
e 

f 

g 
h 

39.61 


0.0 
0.0 
0.0 
0.0 
Trace? 
0.0 
0.0 
0.0 
0.0 
0.0? 
0.0 


4.00 

Trace? 

0.0 

11.6 
7.5 
3.3 
3.7 
4.0 
0.0 
0.6 
Trace? 

48.8 


29.1 
48.8 
20.0 
23.6 
29.8 

6.3 
15.1 
14.5 
10.6 

9.8 
44.0 

3.8 


Imported, eyes (?). 

Imported, eyes normal. 
Do. 
^Domestic, eyes thickly 
/ crowded. 

Do. 
^Imported, eyes thickly 
/ crowded. 

\ Excellent imported, eyes 
/ very regular. 

Imported, large hole. 

Very gassy in press. 



12 



STUDY OF GASES OF EMMENTAL CHEESE. 



The analyses of the gas collected by Method I are given in Table 1, 
and of that collected by Method II in Table 2. All volumes are for 
0° C. and 760 mm. When the gases were collected from a cheese pro- 
cured at the market, a sufficiently large slice was purchased to pre- 
vent undue exposure of the eyes, and this was carried immediately 
the short distance to the laboratory, and the gas at once collected. 
In most cases the shaft punctured or grazed more than one eye, so 
that the analysis gives the true average for several eyes. 



Table 2.— An 



of gas collected by pumping from Swiss (Emmental) 
Method II. 



No of 


Time 
pump- 
ing. 


Total 
gas col- 
lected. 


Weight 
of cheese 
evacua- 
ted. 


Amount 
of gas 

per 100 
grams of 

cheese. 


Composition. 




cheese. 


C0 2 . 


o 2 . 


H 2 . 


N 2 . 


Description of cheese. 


3 


Hours. 
20 

20 ~ 

20 " 


C.c. 
2.36 

2.31 
6.41 
3.20 
13.60 


Grams. 


C.c. 


Per 
cent. 
76.3 

77.5 
80.8 
50.6 
84.5 


Per 

cent. 
1.7 

2.6 
2.0 
1.0 
2.2 


Per 

cent. 
0.0 

0.0 
0.0 
0.0 
0.0 


Per 

cent. 
22.0 

19.9 
17.2 
48.4 
13.3 


Almost blind. Several small 


39-45 






holes, either pinholes or in- 
hibited eyes. 
Do. 


39-11-2 






Do. 


46-4-1 

W2 


50 
53 


6.40 

25.7 


Do. 

Fine domestic cheese just be- 
ginning eye development. 



DISCUSSION OF THE ANALYSES. 

If the values obtained in this study of the gases found in the eyes 
of Swiss cheese are compared with the values obtained by Boekhout 
and Ott de Vries 2 for the gases in Edam cheese, it is seen that the 
latter obtained much lower percentages of carbon dioxid and corre- 
spondingly higher percentages of nitrogen. The explanation becomes 
apparent when it is remembered that Boekhout and Ott de Vries 
collected the gas over water, while in this investigation it was collected 
over mercury. The two methods were compared in the case of cheese 
h, as follows: 



Method. 



Collection over mercury 
Collection over mercury 
Collection over water... 



C0 2 . 



Per cent. 
81.2 
80.9 
34.8 



2 , 



Per cent. 

Trace. 

0.6 

1.9 



H 2 . 



Per cent. 
3.7 
4.0 
1.9 



N 2 . 



Per cent. 
15.1 
14.5 
61.4 



This result is what might have been expected, namely, an absorp- 
tion of much carbon dioxid and a little hydrogen by the water, and, 
in return, an increase in the amount of oxygen as well as an increase 
in percentage of nitrogen. Boekhout and Ott de Vries have them- 
selves called attention to this, and claim only qualitative value for 
their results. The types of holes from which they isolated gas were 
small cracks corresponding to the Emmental "riszler," small round 
holes, and large cracks termed "knijpers." 



DISCUSSION OF ANALYSES. 13 

Qualitatively the composition of the gases was the same, namely, 
carbon dioxid, hydrogen, nitrogen, and oxygen. Of these they elimi- 
nated oxygen as due to contamination. In the case of the "knij- 
pers, 7 ' or large cracks, 52 to 249 c. c. of gas were collected instead of 
5 to 22 c. c. as in the case of the smaller holes. Assuming that the 
same volume of water was used, we would expect a truer value to be 
obtained for the analysis of the larger volumes, in which case the 
attention is struck by the large percentage of hydrogen. The signifi- 
cance of this will become apparent when the results on Emmental 
cheese have been assembled. 

It is clear from the analyses of gas found in Emmental cheese 
that carbon dioxid and nitrogen are the chief constituents of the gas 
found in normal eyes. The oxygen in most cases is hardly more than 
would be expected to come from the minute bubbles or surface 
layers which adhere to the glass walls of the apparatus. To what 
gas the contraction after explosion with oxygen is to be ascribed is a 
difficult question to settle. In some cases, where the contraction 
was sufficiently large to justify further absorption with potassium 
hydroxid, the absence of any further contraction in volume justifies 
the conclusion that the combustible gas was chiefly hydrogen. In 
other cases the small contraction might have been due to any one of 
a number of gaseous combustions. 

For further information it was decided to examine specimens of gas 
spectroscopically. The gas freed from carbon dioxid and possible 
oxygen was passed over phosphorus pentoxid into a dry, exhausted 
Plucker tube. The discharge of an induction coil was then passed 
between aluminum terminals, and the spectrum observed with a 
prism spectroscope. At the same time comparison was made with the 
spectrum of a similar tube containing pure hydrogen. Minute traces 
of hydrogen are to be expected when metal terminals are used, but, 
with the low resolving power of the spectroscope employed, the nitro- 
gen spectrum so obscured the possibly present red line of hydrogen 
that it was not observed with specimens of pure nitrogen. A known 
sample of nitrogen containing about 0.05 per cent of hydrogen gave 
a brilliant hydrogen spectrum, whose intensity could be made more 
sharp at the expense of the nitrogen spectrum by suitable varying of 
the pressure. 15 The recognition of 0.05 per cent of hydrogen was 
therefore assured. 

A small experimental cheese, which had begun an apparently nor- 
mal eye formation and then ceased entirely, was pumped out by 
Method II and its gas submitted to spectroscopic examination. 
Slight evidences of hydrogen were observed. Samples of gas taken 
from cheeses which yielded 3 per cent of combustible gas gave very 
brilliant evidences of hydrogen. 



14 



STUDY OF GASES OF EMMENTAL CHEESE. 



In samples of gas taken from the normal eyes of two cheeses pur- 
chased on the market no hydrogen line was observed, nor was the 
hydrogen spectrum observed in the gases of a normal cheese evolved 
during the period of its maximum eye formation. 

These results, though not extensive, 
are sufficient to show that hydrogen 
plays no r61e in the formation of normal 
eyes, provided we assume that any hy- 
drogen formed has not escaped collection 
by rapidly diffusing through the cheese. 
To make sure of this point the following 
experiments were conducted: 

Two cheeses purchased in Wisconsin 
were found to be developing normal eyes. 
These eyes, though too thickly scattered 
for the modern market standard, would 
have been declared typical some years 
ago. When each cheese was apparently 
at the height of its eye formation, plugs 
were taken, and introduced into the tube 
A, figure 3, without that part illustrated 
at the side and lettered G, F, and E. To 
guard as far as possible against infection 
in transference the trier was flamed, and 
the tube was sterilized at 170° C, with 
cotton plugs at B and C. After intro- 
ducing the plugs of cheese they were fol- 
lowed by the flamed cotton plug and then 
a rubber stopper dipped in hot rubber 
cement. The stopper was forced in and 
held in place till the cement a had cooled, 
when several layers of the same cement 
were added to the exterior. This made 
a thoroughly gas-tight seal. The capil- 
lary end was now attached to the mercury 
pump by means of securely tied rubber 
tubing completely covered with a mercury seal. Then the tube was 
exhausted. 

Forty-six grams from one of the Wisconsin cheeses were exhausted 
for two hours, during which time it continued to give off small 
quantities of gas. The pressure was finally reduced to 2 mm. (meas- 
ured on a McLeod gauge). The stopcock T> was then closed, and the 

a The cement was made by heating rosin several days with as much fine-grade rubber as it would dissolve. 
Dr. Nutting, of the Bureau of Standards, who kindly furnished the receipt, stated that he had used this 
cement in refined vacuum work with entire satisfaction. 




Fig. 3.— Apparatus for studying the ab- 
sorption of oxygen by cheese. 



DISCUSSION OF ANALYSES. 15 

tube allowed to remain in connection with the pump overnight. 
The next morning the pumping was resumed, and a pressure of 2 mm. 
again obtained. The gas which had collected overnight amounted 
to 7.23 c. c, N. T. P. Its analysis follows: 

C.c. 

Original volume 7. 23 

Residue after absorption with KOH 17 

C0 2 7.05 

Oxygen added up to a 2. 28 

Volume after combustion with heated platinum spiral 2. 26 

Contraction 02 

The tube was then sealed off in a blowpipe at the constriction H 
and kept for six days at 25° C. To collect the gas from this sealed 
tube the following method was used. The capillary tip of the seal 
was scratched with a diamond, and then pushed up into the tube 
leading from the pump as at C, figure 3. Connection was made with 
a rubber tube securely tied and covered with a mercury seal. Having 
exhausted the pump up to the tip of the seal, the tube was turned 
slightly and sharply. The tip was broken at the scratch, and com- 
munication established between A and the pump. 

The gas thus collected at the end of six days amounted to 10.12 
c. c. The tube was allowed to stand connected with the pump over- 
night, after which an additional 2.75 c. c. of gas were collected. 

These two volumes were united and analyzed 99.3 per cent carbon 
dioxid. The residue was hardly sufficient to justify further analysis. 
It was made, however, and a minute contraction observed, which was 
hardly more than the experimental errors of transference. 

Forty-five grams of the second Wisconsin cheese submitted to the 
same procedure as described above gave the following data: 

C.C. 

Gas collected on first standing overnight 11. 48 

Residue after absorption with KOH 22 

C0 2 11. 26 

Residue after absorption with phosphorus .22 

Oxygen added up to a 2. 41 

Volume after combustion with heated platinum spiral 2. 39 

Tube sealed off and incubated six days at 25° C. 

C.c. 

Gas collected after 6 days 9. 29 

Residue after absorption with KOH 16 

C0 2 9. 13 

Oxygen added up to a 4. 91 

Volume after combustion with hot platinum spiral 4. 85 

Gas collected after again standing overnight 5. 33 

Residue after absorption with KOH Trace. 

a This comparatively large volume was made necessary because of the disadvantageous form of the 
Dennis-Hopkins pipette used. 



16 STUDY OF GASES OF EMMENTAL CHEESE. 

In the above analyses the contraction due to combustion was so 
small that further analyses to determine the products of combustion 
were impracticable. Nor was it necessary, for, even if the contraction 
were due to but one gas, for example hydrogen, the amount was such 
that this gas may be said to be without significance in the formation 
of eyes. Doubtless the contraction was in reality due to volatile 
organic bodies. The above experiments show that when all the gas 
from an actively gas-producing region is collected no significant 
amount of hydrogen is found, and thereby the contention is refuted 
that, in the analysis of gas in the eyes, hydrogen escaped detection 
because of its rapid diffusion out through the cheese. 

Pains were taken in these studies to make a strenuous hunt for 
hydrogen for the following reason: In Emmental cheese there is 
what Duclaux has termed the "initial fermentation" during which 
the sugar inclosed in the curd undergoes bacterial decomposition. 
Several of the earlier workers on this cheese thought it was the gaseous 
fermentation of this sugar which caused the development of eyes. If 
so, one would expect to find the gas composed of a large percentage of 
hydrogen, since hydrogen is a characteristic product in the fermenta- 
tion of sugars by bacteria. This deduction is of course not rigid, but, 
from our present knowledge of the gaseous fermentation of sugars by 
bacteria, it is highly probable. 

Jensen u in 1898 pointed out clearly that the gaseous fermenta- 
tion of sugar must not be looked upon as in any way directly connected 
with the production of normal eyes in Emmental cheese. He 
found no trace of sugar in a cheese five days old, although the normal 
eye formation had not yet begun. This confirms the analyses made 
by various authors. Jensen cited Klenze 13 as stating that the sugar 
disappears in 48 hours. But, while the sugar disappears rapidly, 
normal eyes seldom begin to develop before the eighth day, and reach 
the height of their development long after every trace of sugar has 
disappeared. 

These facts alone demonstrate that the eye formation does not 
depend upon the presence of sugar. Additional reason for so believ- 
ing is found in the results herein, in so far as the absence of hydrogen 
in the gas indicates an absence of gaseous sugar fermentation. 

But it also follows from this reasoning that when a gaseous fermen- 
tation occurs while sugar is still present in the cheese, hydrogen is to 
be expected. Such a fermentation frequently occurs while the cheese 
is in press. Fortunately a cheese was obtained (No. 39-61) which was 
known to have given marked signs of gas while under press. From 
this cheese gas was collected by the previously described Method I, 
with the following analysis : 

Total volume of gas collected Cubic centimeters. . 4. 96 

Residue after absorption with KOH do 2. 61 

C0 2 do. . . . 2. 35 

Residue after absorption with phosphorus do 2. 60 



DISCUSSION OF ANALYSES. 17 

Oxygen added up to Cubic centimeters. . 6. 14 

Volume after combustion with platinum spiral do 2. 51 

Contraction do 3. 63 

Residue after absorption with KOH do 2. 51 

Hydrogen per cent. . 48. 80 

Upon attempting to make a second puncture the mercury broke 
through into the hole previously made. The cheese was then opened, 
and found to be so spongy that the walls separating the individual 
cells were very thin — too thin to withstand the weight of mercury. 

To obtain a second sample of gas for confirmatory analysis recourse 
was had to Method II of collecting gas, previously described. A high 
percentage of hydrogen was again found. 

In the further study of this case 52 grams of the cheese were intro- 
duced into the vacuum tube described on page 14 and evacuated to 
1 mm. pressure. There collected overnight 7.84 c. c. of gas. 

Analysis : 

Total volume 7. 84 

Residue after absorption with KOH 28 

Residue after absorption with phosphorus 27 

Oxygen added up to 1. 08 

Volume after combustion with platinum spiral 99 

Contraction 09 

The tube was then sealed off and kept nine days at 25° C. Upon 
opening it and pumping out the gas by the method previously 
described 7.49 c. c. of gas were collected. The residue after absorp- 
tion with potassium hydroxid was only 0.07 c. c. 

It is therefore apparent that the production of hydrogen, which 
was very active while the cheese was in press, had soon ceased, pre- 
sumably with the disappearance of the sugar. 

The occasional occurrence of hydrogen in small percentages, as 
shown in the table, generally accompanied eyes which in the writer's 
judgment were not typically normal. They were either crowded and 
distorted or associated with numerous pinholes. It is not, perhaps, 
incorrect to say that in all probability there had occurred in these 
cases a slight initial gaseous fermentation of the sugar, with the pro- 
duction of hydrogen which lingered to contaminate the gas of the 
normal fermentation. 

An extremely interesting observation was made in the case of cheese 
i. (See Table 1, p. 11.) This was an excellent imported cheese with 
large and perfectly rounded eyes, well spaced in a body of fine texture 
and flavor. In the first analysis of the gas from these eyes no trace 
of a combustible gas was found. The second analysis gave 0.6 per 
cent of hydrogen. Upon exposing the eyes punctured it was 
observed that a slight crack extended to within a centimeter of one of 
the eyes punctured on the second collection. This crack was found 
to lead directly to a hole some 2 cm. in diameter, the irregular and 
apparently corroded walls of which proclaimed it distinctly abnormal. 



18 STUDY OF GASES OF EMMENTAL CHEESE. 

It is of interest to note that in the case of cheese j, gas was obtained 
from a hole the size of one's fist, and that this contained practically 
no hydrogen. The appearance of this hole was that of a strictly 
normal eye except in size. 

It was hoped that the gas of a typical "blow hole" could be ob- 
tained. For this purpose a cheese containing such a hole was pur- 
chased in Wisconsin. When it arrived at the laboratory it was found 
that the cheesemaker had punctured it. . 

From the results obtained it is clear that there are at least two 
distinct t}^pes of gas formation. a The one is highly detrimental, and 
is accompanied with hydrogen; the other is that demanded in a good 
Emmental cheese. One is dependent upon the presence of sugar; 
the other occurs in the absence of sugar, 

The presence of hydrogen in considerable quantities in the gas iso- 
lated from Edam cheese by Boekhout and Ott de Vries is very sug- 
gestive of a gaseous fermentation of sugar, and to this Jensen n has 
ascribed the formation of gas holes in Edam cheese. 

At this point it may be well to call attention to a source of error 
overlooked by various investigators in their attempts to establish 
the cause of any particular gas formation in cheese. Frequent exam- 
ples are to be found in which gas production by bacteria in milk is 
interpreted to mean that these bacteria can produce gas in cheese. 
Although this may frequently be true, it must nevertheless be remem- 
bered that the two media differ not only in chemical constitution but 
also vary greatly in physical chemical condition. 

Baumann, 3 for instance, attributed the formation of eyes in hard 
cheeses to Bacillus diatrypeticus casei. From an experiment in 
which this bacillus produced in milk gas containing 63 per cent of 
carbon dioxid and the remainder almost entirely hydrogen, Baumann 
concluded that the gas of normal as well as faulty eyes is carbon 
dioxid and hydrogen. The error of attributing the reactions of a 
bacillus when cultivated in milk, which contains sugar, to cheese, 
which after the initial fermentation contains no sugar, is so evident, 
and the error in stating that the gas of normal eyes contains hydrogen, 
without having first analyzed this gas, is so evident, that Baumann's 
conclusions might be left unnoticed at this late date were they not 
typical of several found in the more recent literature. 

ABSORPTION OF OXYGEN. 

In all the analyses no appreciable amount of oxygen was found. 
The presence of large percentages of nitrogen with this absence of 
oxygen raises the question, Does air diffuse into the cheese with ab- 
sorption of oxygen ? Evidence of an active absorption of oxygen was 

a This does not preclude there being a number of distinct fermentations or reactions of either type. 



ABSOKPTION" OF OXYGEN. 19 

accidentally obtained. In attempting to study the gases produced 
in sealed tubes a faulty form of tube was first used, which evidently 
leaked. On attempting to exhaust ; the lowest pressure which could 
be obtained was 3.6 mm. It was soon ascertained that there was no 
leak in the pump, but a leak in the tube was suspected. The tube 
was left connected with the pump (connecting stopcock closed) over 
night. The next morning 37.20 c. c. of gas was pumped out. The first 
portion of 19.15 c. c. gave 4.57 c. c. of carbon dioxid and 2.21 c. c. of 
oxygen. The residue was lost but was considered to be nitrogen. The 
second portion was then pumped out, and of the 18.05 c. c. thus col- 
lected there were 4.85 c. c. of carbon dioxid, 1.45 c. c. of oxygen, and 
the residue entirely nitrogen. The total oxygen amounted to 3.66 c. c, 
which, had it come by leakage, would have indicated an entrance of 
13.7 c. c. of nitrogen. There was actually found 24.12 c. c. of nitrogen. 
This leaves 10.42 c. c. of nitrogen to be accounted for. The carbon 

10 42 
dioxid amounted to only 9.42 c. c. and, since the ratio ' An is much 
J 9.42 

larger than that obtained in other similar pumpings where no leak 
occurred, it was suspected that oxygen had been absorbed. 

To definitely determine this the apparatus shown in figure 3 was 
used. With plugs of cotton at B, C, and in the bend above G, the 
tube was sterilized at 170° C. Then 28.5 grams from one of the Wis- 
consin cheeses were carefully taken with trier and spatula flamed to 
prevent contamination as far as possible, and the plugs introduced 
into A and sealed- in as previously described. Mercury was drawn up 
into the tube E until it had just passed the stopcock F. After at- 
tachment had been made to the pump the whole was evacuated 5 
hours and finally at a pressure of 1.2 mm. the capillary at H was sealed 
off in a blowpipe flame. There was introduced into E 7.47 c. c. N. 
T. P. of oxygen from a tank. At the same time a sample of the same 
gas was taken for analysis, and found to contain 98.1 per cent of 
oxygen. Upon opening the cock F atmospheric pressure forced the 
gas over into the tube A. The mercury behind this gas was allowed 
to rise until it had entered the capillary G. As close to this mercury 
as was possible G was then fused off with a blowpipe. There was 
left of the 7.47 c. c. introduced only a small bubble in the capillary, 
and this at reduced pressure. After 6 days at 25° C. the tube was 
opened by the usual method and the gas was pumped out and ana- 
lyzed, with the following result: 

c. e. 

Total volume of gas collected 11. 90 

Carbon dioxid 10. 96 

Oxygen 53 

Residue, all nitrogen 41 

From the percentage composition of the 7.47 c. c. of gas added 
at the beginning of the experiment it is known that 7.33 c. c. of 



20 



STUDY OF GASES OF EMMENTAL CHEESE. 



oxygen was added. At the end of the experiment there remained 

only 0.53 c. c. of oxygen. There must, therefore, have been 6.80 c. c. 

of oxygen absorbed, or 0.239 c. c. per gram of cheese. 

Such an active absorption of oxygen lends itself to the argument 

that the nitrogen of the eyes found its way there by the diffusion in of 
air. But, before such an argument can be con- 
sidered valid, the following points must be deter- 
mined : First, to what extent is cheese permeable 
to gases in general and nitrogen in particular ? 
Second, how much of the nitrogen present is due 
to nitrogen dissolved in the cheese at the time of 
manufacture ? Third, what evidences are there 
to show that the nitrogen does not arise in situ 
from bacterial or chemical reactions ? 



F 



K=0 




THE PERMEABILITY OF CHEESE TO GASES. 

After various unsuccessful efforts to make an 
impermeable adhesive that would stick to 
cheese, and so enable a slice to be sealed into 
a diffusion apparatus, the following device was 
made (fig. 4) : 

At B a membrane of plaster of Paris was 
formed whose strength was reenf orced by a per- 
forated brass plate not shown in the diagram. 
This membrane was desiccated until its perme- 
ability was high, that of transfusion. 10 

Most of the air was forced out of D through 
the membrane and E by raising the mercury. 
A carefully taken disk of cheese was then 
placed on the plaster of Paris bed. It was 
gently held there while it was completely cov- 
ered with mercury. Then, by lowering F, the 
space in D was left under greatly reduced pres- 
sure. This caused such a difference in pressure 
between the upper and lower surfaces of the 
cheese that the disk was held firmly against the 
plaster bed, and the surrounding mercury was 
unable to float it. Preliminary experiments 
showed that no mercury crept between the disk and the plaster, 
and that the plaster did not become clogged with mercury or cheese. 
After partial vacuum had been produced in D a few moments elapsed 
before the gas retained in the plaster came to equilibrium. When 
this was reached the mercury was carefully withdrawn from the top 
of the disk of cheese until the surface was exposed. The mercury 



Fig. 4. — Device for ascertain- 
ing permeability of cheese to 



PEEMEABILITY OF CHEESE TO GASES. 



21 



left at the side prevented entrance of gas there, so that the only 
path between the chambers A and D by which gas could enter D was 
through the cheese. 

The disks of cheese used were about 1 cm. in diameter and 2 to 
2.5 mm. thick. They were taken from sound portions of freshly cut 
cheese by means of a cork borer, and carefully sectioned with a sharp 
razor. Every precaution was used in cutting and handling to pre- 
vent distortion and breaking of the texture. In one case the exposed 
surface was that of an eye. The gas whose diffusion it was desired to 
study was flooded into the chamber A. With both air and carbon 
dioxid there was apparently no diffusion during an hour, even though 
the pressure in D was reduced as much as possible. Longer experi- 
ments were not practicable, because a continuous watch had to be 
kept to see that no bubble of air entered through the rubber connecting 
tube between D and F and altered the volume in D. With a trap to 
prevent such a source of error the same impermeability for air was 
observed during an experiment lasting several days. 

This result was so remarkable that it was tested further in the 
following manner: Instead of the parts E, D, F (fig. 4), a glass tube 
led from B to a mercury pump. With the cheese slab C covered with 
mercury the pump was operated till the lowest vacuum which could 
be obtained was reached. By reason of the gas being given off by the 
cheese, this was of course not so high a vacuum as the pump can 
produce. When the vacuum was considered sufficient the pump was 
allowed to rest in order to discover leakage, and, if there were none, 
to allow the residual gas to distribute itself so that a reliable reading 
on the McLeod gauge could be made. Then the mercury was care- 
fully withdrawn from the top of the cheese, leaving its upper surface 
exposed. Entrance of gas could now be detected by the McLeod 
gauge. An experiment is given in detail below: 

[Disk of cheese 7 mm. diameter, 2.5 mm. thick, taken 10.50 a. m., Dec. 19, 1911, 15 mm. from the nearest rind.] 



Time of 
reading. 



Pres- 



Apparatus exhausted, and, with cheese covered with mercury, pump pressure at 

Pump resting 

Do 

Increase in pressure assumed to be due to gas evolved from cheese. 

After 7 minutes pumping 

Cheese exposed to air 

• Do 

.Do 

Do 

Do 

After 15 minutes pumping 

Cheese exposed to CO2 

Do 

After 5 minutes pumping 

Cheese exposed to H2 

Do 

After 15 minutes pumping 

Cheese left overnight exposed to air 



a. to. 
11.14 
11.25 
11.30 

11.37 
11.40 
p. to. 
12.15 
1.15 
1.50 
2.15 
2.30 
3.10 
3.30 
3.35 
4.10 
4.30 
4.45 
a. to. 



Mm. 
0.075 
.140 
.150 

.070 
.070 

.150 
.270 
.320 
.350 
.025 
.060 
.090 
.025 
.050 
.065 
.010 

.430 



22 STUDY OF GASES OF EMMENTAL CHEESE. 

When the disk of cheese and the mercury were removed air entered 
rapidly, showing that the plaster had not become plugged. Further- 
more, there was no evidence of mercury having crept between the 
cheese and the plaster. It is not claimed that all the above listed 
readings on the McLeod are very accurate, since the readings were 
sometimes made before equilibrium was obtained. All that was 
desired was the order of magnitude. Since the variation in tempera- 
ture during the experiment was only between the extremes 17° C. 
and 19° C. and since the volume of the pump, gauge, and diffusion 
apparatus was found to be 159 c. c, we may calculate from pressures 
the approximate amount of gas which had apparently diffused through 
the cheese. This amounted to about 0.09 c. c. during the first 5 hours 
and 0.09 c. c. during the last 17 hours. 

Allowing nothing for possible small leaks, which were difficult to 
avoid in the delicate manipulations required, the observed volume of 
gas indicates a very remarkable impermeability. Practically the 
same result was obtained with a disk of Cheddar cheese and other 
samples of Swiss cheese. 

The question at once arises, How to explain the evolution of carbon 
dioxid which there is every reason to suppose does diffuse from cheese ? 
Van Slyke and Hart 17 found that a normal Cheddar cheese evolved 
during 32 weeks 15.099 grams of carbon dioxid. Since they took 
care to exclude surface growths of molds, it seems highly improbable 
that this amount of carbon dioxid could have come to any great 
extent from the surface layers alone. It must have diffused from 
the interior of the cheese into the surrounding bell jar. 

The following explanation will doubtless be found reasonable: 
Becquerel 4 found that when the tegument of peas was mounted at 
the end of a barometer tube, and a partial vacuum of 5 to 10 mm. 
obtained upon the one side, with atmospheric pressure on the other, 
the tegument was impermeable to gas when dry, although permeable 
when moist. In so far as the tegument of peas and a disk of cheese 
are both colloidal they may be compared. In the present experi- 
ments the disks of cheese dried considerably both from exposure to 
gases of low vapor content on the one side and the moisture free 
vacuum on the other. By analogy with BecquereFs experiments 
one would expect to find the dry cheese more or less impermeable. 
Keference to the experiment detailed on page 21 will indeed show that 
the permeability decreased as the time of the experiment increased, 
or, in other terms, as the cheese became drier. Furthermore, in 
an experiment in which the exposed surface of the cheese was kept 
exposed to carbon dioxid, which was saturated with vapor, 1.04 c. c. 
of gas was found to have passed through in 5 hours; ten times as 
much as in the experiment with drying cheese. 



PEKMEABILITY OF CHEESE TO GASES. 23 

It therefore seems probable that the permeability of cheese to gases 
is due to the diffusion of dissolved gases, and that as the free solvent 
becomes more and more attenuated the gas is more and more unable 
to find its way through the gel. 

Since in Emmental cheese a more or less dry rind is produced, it 
seems probable that little air can diffuse into the cheese. And from 
the fact that in the manufacture of Cheddar a less dry rind as well as 
a more open texture is produced, it seems probable that escape of 
carbon dioxid more easily occurs in tins type than in the Swiss type 
of cheese. 

It must be remembered, however, that the above experiments only 
cover a very limited time, and that, even were the permeability as 
low as the experiments seem to show, there is still the possibility that 
nitrogen may make its way slowly through the gel during the long 
period of ripening. Possibly more extensive investigation would 
reveal that the larger percentages of nitrogen found in the eyes of 
some cheeses are proportional to the age of the cheeses. Neverthe- 
less this penetration can only take place slowly. 

The fact that penetration of air is so slow, together with the 
avidity with which oxygen is absorbed, only tends to emphasize the 
completeness of the anaerobiosis in the interior of the cheese, a con- 
dition which Troili-Peterson 16 found necessary for the best develop- 
ment of the propionic bacteria. 

These experiments on the permeability of cheese to gases make it 
evident that in pumping the gases from plugs of cheese we should ex- 
pect the gas to be slowly evolved. Such was, indeed, found to be the 
case. The reason for this was not fully appreciated at the time the 
pumpings were made, and it is very doubtful if all the occluded gas 
was completely exhausted even after 20 hours exposure to high 
vacuum. Reference to the experiment with plugs of cheese kept 6 
days in vacuo (p. 15) reveals the interesting fact that the amount of 
gas evolved per gram of cheese was dependent more upon the state 
of the vacuum than upon time. This is illustrated in the following 
statement, in which the figures represent cubic centimeters of gas 
evolved per gram of cheese per hour: 



First 18 hours 

Succeeding 6 days. 
Last 18 hour's 



0.0087 
.0015 
.0033 



0.0042 
.0014 



During the middle period, of course, the tubes were sealed, and the 
evolved gas increased the pressure. Evidently, then, the higher the 
vacuum to which the sample was subjected the more rapidly was the 
gas evolved, indicating that a considerable proportion of the gas was 



24 



STUDY OF GASES OF EMMENTAL CHEESE. 




dissolved or occluded gas rather than that formed during the time 
of the experiment. 

It may also be true that there is loose combination of carbon 
dioxid with inorganic salts, or with calcium and ammo bodies, as in 
the carbo-amino reaction, and that the stability of these compounds 
is a function of the imposed pressure. 

NITROGEN DISSOLVED IN CURD. 

Let us now consider how much of the 
nitrogen found in the eyes is attributable 
to nitrogen occluded in the original curd. 
One would expect the curd to be well 
aerated by the vigorous stirring it gets 
during the process of manufacture. Mar- 
shall 14 has shown that aerated milk con- 
tains considerable quantities of nitrogen, 
but, unfortunately for the purposes de- 
sired, his data are only expressed in per- 
centage composition and not very defi- 
nitely in cubic centimeters of gas per 
cubic centimeter of milk. 

A rough approximation of the amount 
of nitrogen occluded in the curd was 
obtained in the following way: A liter 
of milk was treated as in the process of 
making Swiss cheese. When the curd 
had reached the stage when it was suit- 
able to hoop, the greater part of the 
whey was decanted, and then the re- 
sidual whey and curd were poured care- 
fully into the glass cylinder A, figure 5 
(inverted). As the curd settled, the 
overlying whey was drawn off and more 
of the mixture poured in. This was re- 
peated until the tube was filled with curd 
grains completely surrounded by t whey. 
The rubber stopper was then forced in. 
The tube was next inverted to the posi- 
tion shown in the figure, and the mer- 
cury seal stopcock B was opened to re- 
lieve the pressure. The rubber stopper was then forced farther in, 
and the whey displaced by it escaped into C. By covering the 
stoppered end of the tube with rubber-rosin cement and keeping 
it under mercury, it was made perfectly gas tight. The cock B was 
then closed, and, after the surplus whey in C had been drained out, 
the apparatus was connected to the vacuum pump in the usual way. 




GLASS 



Fig. 5.— Apparatus for determining 
amount of nitrogen In curd. 



NITROGEN DISSOLVED IN CURD. 25 

When the pump and chamber C were completely exhausted, the 
cock B was opened. It was found that the gas expanding in A 
drove the whey almost completely up through the interstices of the 
curd and into C. 

An interesting point was observed. Comparatively little of the 
gas came from the whey, while the major portion came from the curd 
particles. Since a separation of whey and curd was accomplished, 
it could not have been true that the gas evolved from the curd par- 
ticles originated in the whey, using curd particles as nuclei for the 
formation of bubbles. Furthermore, there was comparatively little 
frothing of the whey in C, most of the gas collected having bubbled 
through C from A. Examination of curd particles will show why 
this is so; for they have adhering to them minute bubbles, apparently 
froth taken up during the stirring. It is quite evident that the col- 
umn of whey in C through which the gas had to make its way pre- 
vented a very complete exhaustion. Since the pumping was con- 
tinued several hours and the tube then allowed to stand overnight 
before the final pumping, this error was reduced to some extent. If 
occasion arises to repeat these experiments this error will be avoided. 

By the method described, 1.35 c. c. of gas was collected in one 
instance and 0.86 c. c. in another. Of this, there was 0.58 c. c. of 
nitrogen in one case and 0.39 c. c. in the other; average, 0.5 c. c. 
The curd was roughly estimated to represent 20 grams of cheese. 
Consequently there would be approximately 2.5 c. c. of nitrogen 
per 100 grams of cheese. How this nitrogen would partition itself 
between the body of the cheese and an eye is a question whose solu- 
tion would be mere guesswork without further data. 

While the 2.5 c. c. per 100 grams of cheese is a mere approximation, 
and a figure which would vary not only with the extent to which the 
curd is stirred, but also with the form of the curd particles and their 
ability to absorb foam, nevertheless it is sufficiently accurate to 
show that a large part of the free nitrogen found in cheese comes from 
occluded air. 

DOES NITROGEN ORIGINATE IN SITU? 

The question of whether any of the nitrogen found in the eyes is 
set free in situ is a difficult one to answer, and one which can not be 
definitely answered without further research. From the following 
considerations, however, it is highly probable that it is not produced 
during the course of that reaction which furnishes the gas to distend 
the eyes. In those experiments in which samples from a cheese at 
the period of its maximum eye formation were held in vacuo, the 
nitrogen in the evolved gas steadily and rapidly declined in per- 
centage, finally reaching almost nothing. This indicates that the 
nitrogen collected was simply that dissolved in the cheese, and as 



26 STUDY OF GASES OF EMMENTAL CHEESE. 

this was removed there was no evolution of free nitrogen to take its 
place, such as occurred in the case of the carbon dioxid. 

RELATION BETWEEN CARBON DIOXID AND VOLATILE ACIDS. 

The results of the whole investigation show clearly that the only 
gas which plays an important role in the formation of normal eyes is 
carbon dioxid. This is in entire harmony with the assumption which 
has heretofore been accepted as a fact by various authors. 

It remains to be seen whether there is any quantitative relation 
between the amount of carbon dioxid evolved and that called for by 
the process to which Von Freudenreich and Jensen ascribe the forma- 
tion of eyes. 

A study of the volatile fatty acids of Emmental cheese by Jensen 12 
disclosed the fact that they are chiefly propionic and acetic, and that 
often the ratio of these approximates 2:1. 

Fitz 7 had previously shown that certain bacteria are capable of 
producing this ratio of propionic and acetic acids from lactic acid, 
and he ascribed to their action the equation: 

3C 3 H 6 3 =2C 3 H 6 2 +C 2 H 4 2 +C0 2 +H 2 
lactic propionic acetic 

Subsequently Von Freudenreich and Jensen 8 isolated from Emmen- 
tal cheese an organism which did ferment lactates according to the 
above equation of Fitz, and whose introduction into cheese was fol- 
lowed by an eye formation of which it was thought to be the cause. 

The conclusion seems evident that here is an organism to whose 
action may be attributed the formation of normal eyes. 

The evidence is undoubtedly the clearest that has yet been presented. 
There are, however, one or two points. which will bear inspection 
before the theory can be accepted as a full explanation. 

According to the equation of Fitz three molecules of volatile fatty 
acids are accompanied by the liberation of one molecule of carbon 
dioxid. Consequently it can be shown that a titer of 1 c. c. of 
tenth-normal alkali for these volatile fatty acids should indicate the 
liberation of 0.74 c. c. of carbon dioxid (N. T. P.)._If, then, it is 
found that the volatile acids from 100 grams of cheese neutralize 100 
c. c. of tenth-normal alkali, and it is assumed that these acids are 
acetic and propionic in the ratio in which they occur in Fitz's equa- 
tion, we would have liberated 74 c. c. of carbon dioxid per 100 grams 
of cheese. 

This amount of gas is considerably more than is required to fill the 
eyes, but the question remains how much is to be found in the body 
of the cheese itself. 

Reference to the experiments described on page 15 shows that at 
an age of 55 days 37.2 c. c. of carbon dioxid per 100 grams of cheese 



KELATION" BETWEEN" CAKBON" DIOXID AND VOLATILE ACIDS. 27 

were collected after the cheese has been held in vacuo one week. At 
the time of the experiment it was thought that this gas was produced 
during that week. After the study which shows how impermeable 
cheese is, this view had to be modified, for, even after considerable 
pumping, an appreciable quantity of gas must have remained and 
appeared as " evolved" gas at the end of the week. In order to make 
a better estimation of the dissolved gas, plugs of this same cheese (at 
an age of 4 months) were sliced into thin disks to facilitate the removal 
of dissolved gas, and introduced into a tube. They were sealed in 
with the usual rubber stopper and rubber-rosin cement, and the tube 
joined to the mercury pump. After evacuating the pump the con- 
necting cock was opened and the disks of cheese evacuated. The air 
surrounding them in the tube was of course pumped out too. The 
total gas thus collected after 5 hours continuous pumping contained 
17.05 c. c. of carbon dioxid. The weight of cheese was 42 grams. 
Hence, there were collected 40.6 c. c. of carbon dioxid per 100 grams of 
cheese (19 hours later 0.9 c. c. of carbon dioxid was collected, or 2.1 
c. c. per 100 grams of cheese). 

A duplicate determination gave 46.2 c. c. of carbon dioxid per 100 
grams of cheese (with an additional 1.03 c. c. per 100 grams after 19 
hours). The average for the first 5 hours' pumping was 43.4 c. c. of 
carbon dioxid per 100 grams of cheese, and this we may fairly con- 
sider the quantity occluded at the time the plugs were taken. At 
the same age (4 months) the volatile fatty acids corresponded to 40.9 
c. c. of tenth-normal alkali per 100 grams of cheese. 

Similarly, duplicate determinations of dissolved carbon dioxid and 
volatile acids in an excellent imported cheese (No. i) gave the fol- 
lowing data: Carbon dioxid per 100 grams, 67.8 c. c. and 54.8 c. c, 
average 61.3 c. c. Total volatile fatty acids in cubic centimeters of 
tenth-normal alkali per 100 grams 95.1 and 97.7, average 96.4. 

Assuming that all the volatile fatty acids were produced in strict 
accordance with the equation of Fitz, the amount of these acids in 
the first cheese indicates that there had been liberated 30.6 c. c. of 
carbon dioxid against 43.4 c. c. found occluded; and in the second 
cheese the liberation of 71.3 c. c. of carbon dioxid against 61.3 c. c. 
found occluded. There is a somewhat striking apparent relation- 
ship in this data, and the averages, 51.0 c. c. calculated, against 
52.3 c. c. found, are in such close agreement that they are tempting. 
A little consideration will show, however, that this agreement may be 
only accidental. At the time these analyses were made each of the 
cheeses had probably reached a state of little activity. The volatile 
acids represent almost entirely the total amount produced in the 
interior from which the samples for analyses were taken; while, if 
we are to accept the results on Cheddar cheese by Van Slyke and Hart 
as at all applicable to Emmental, it is certain that a considerable 



28 STUDY OF GASES OP EMMENTAL CHEESE. 

quantity of carbon dioxid must have escaped in the months since 
manufacture. Furthermore, although the actual volume of the eyes 
represents but a small portion of the gas in a given volume of cheese, 
the normal volume of this gas in the eyes leaps into considerable 
significance when it is remembered that it must have been under 
considerable pressure. That it is under pressure was made evident 
in some cases by its vigorous escape when using the puncturing 
apparatus for its collection. 

Unfortunately long delay in obtaining apparatus suitable for a 
study of the gas escaping from cheese, as was done by Van Slyke and 
Hart for Cheddar, have made it impossible to present any data on 
this point. As before mentioned, the data on carbon dioxid evolved 
from plugs of cheeses taken at the height of their gaseous fermenta- 
tion and kept in vacuo a week is complicated by the fact that there 
was probably a slow yielding of dissolved gas from the solid plugs as 
well as the normal production of gas. Two other experiments, how- 
ever, indicate to what extent carbon dioxid was being formed during 
this period of maximum fermentation. 

Portions of cheese W 2 from regions without eyes were carefully 
selected and sealed up in a tube as described on page 14. The eye 
membranes were carefully removed from a large number of eyes 
and similarly treated. The tubes were simultaneously exhausted 
with a Bolt wood pump for several hours. Since in these cases the 
cheese was in a more finely divided state, it is reasonable to assume 
that predissolved gas was pretty thoroughly removed. After exhaus- 
tion, the tubes were sealed off in a blowpipe flame and held at 25° C. 
for seven days. At the end of this period the gas was collected: 

34 grams eye membranes gave 14.95 c. c. of gas, 99.3 per cent of carbon dioxid, 

or 44 c. c. per 100 grams. 
36 grams from regions without eyes gave 10.06 c. c. of gas, 98.2 per cent of carbon 

dioxid, or 28 c. c. per 100 grams. 

From this one pair of experiments it is not advisable to claim con- 
fidently that the eye surfaces always produce the much larger quantity 
of carbon dioxid, although this is plainly evident in the above case. 
The significant fact is that such a large quantity was produced by 
each region in the period of only one week. Of course it may be 
claimed that although the division of the cheese was done in a dust- 
free room and with sterile instruments, and the cheese introduced 
into sterile tubes, yet the long manipulation admitted a heavy 
reinoculation by bacteria, and that these produced a renewed evolu- 
tion of carbon dioxid. Such an argument can not be completely 
refuted, but the probability of a heavy enough infection is small. 
The most likely source of carbon dioxid producing infection was by 
molds, but these could not have grown in the complete anaerobic 
condition in which the cheese quickly found itself. 



KELATIOK BETWEEN" CAKBON DIOXID AND VOLATILE ACIDS. 29 

The following experiment serves to confirm the last. Into a steri- 
lized combustion tube were quickly slipped plugs of cheese taken 
with sterile instruments. Each end of the tube was guarded with 
cotton plugs. Carbon dioxid free air was then passed through, and 
the carbon dioxid evolved from the cheese absorbed in the customary 
train with all due precautions for exact estimation of carbon dioxid. 
In the case of this experiment we would expect a higher amount of 
carbon dioxid, since there would be collected not only the carbon 
dioxid produced, but a large portion of the predissolved carbon 
dioxid. Such was found to be the case. 

One hundred and five grams in plugs taken from cheese W 1 when 
at the height of its fermentation gave: 

First 24 hours, 81.6 c. c. of carbon dioxid. 
Second 24 hours, 66.7 c. c. of carbon dioxid. 
Third 24 hours, 44.5 c. c. of carbon dioxid. 
Fourth 24 hours, 63.7 c. c. of carbon dioxid. 

The increase on the fourth day was thought to be due possibly to 
growth of molds with which Van Slyke and Hart found difficulty in 
their work on Cheddar cheese. The experiment was therefore dis- 
continued, although no growth was visible. 

A final word must be urged against the too liberal use of the equa- 
tion of Fitz. As a terse representation of the probable relation of 
the end products the equation has a legitimate use. As a compre- 
hensive portraiture it is colored with presumption. The literature of 
fermentation is littered with equations, two or three members of 
which are known to stand in certain quantitative relationships, while 
the other members are given values which fit. This stoichiometrical 
adjusting is particularly true of the gaseous products. One has only 
to review the literature on the gas production of B. coli to assure 
himself of the fact. 

While Von Freudenreich and Jensen's use of the Fitz equation has 
been interpreted quite rigidly in the preceding pages, this was done 
simply as a test. From this basis alone one can not reasonably jump 
to a final conclusion; but it must be remembered that the liberal use 
made of the Fitz equation was generous to the theory of Von Freuden- 
reich and Jensen in that all the volatile fatty acids, as determined by 
Jensen's method, were assumed to have been produced in accordance 
with this equation. 

From a comprehensive view of the matter it appears to be quite 
evident that the theory of Von Freudenreich and Jensen is not 
capable of accounting for all the carbon dioxid produced. Indeed, 
it is not necessary nor expected that it should, but we have reached a 
point where it has become advisable to distinguish between a primary 
and a secondary cause of eye formation, and to at least define clearly 
what we mean when we attribute to any organism or to any reaction 
the function of forming eyes. 



30 STUDY OF GASES OP EMMENTAL CHEESE. 

Suppose that the propionic bacteria are active, but that they are 
never sufficiently localized to concentrate carbon dioxid rapidly enough 
at one point to produce an eye. In this case the gas would be more 
or less evenly produced throughout the body of the cheese. Now 
this state of more or less complete saturation of the body with car- 
bon dioxid is exactly the condition necessary for the most advan- 
tageous eye formation by any other reaction which may follow, else 
the gas evolved at a point would be largely absorbed and its inflating 
energy dissipated. Of course it can be said that this saturation 
proceeds from the point where the eye is formed, and that the delay 
observed before an eye commences to grow represents the time 
necessary to effect this saturation. 

This, however, is merely presenting the other horn of a dilemma 
from which escape is possible only when the localization of the propi- 
onic bacteria is conclusively demonstrated. Gorini 9 has contended 
that the localization of colonies may often be of as great importance 
as their isolation; and it is interesting to note that he found no cor- 
relation between the colonies which stained on his sections of Grana 
cheese and the gas bubbles. 

If, then, we distinguish between a " saturating" gas production 
and an " inflating" gas production, we will have at least defined a 
possibility which must be squarely met, and a hypothesis which may 
lead to a differentiation between a primary and a secondary cause of 
eye growth. 

The favorable results obtained with cheese inoculated with pro- 
pionic bacteria indicate that they may play an important role. But 
is this role primary or secondary ? Is it a strictly localized action or 
is it simply the provision of that saturation without which some 
primary and strictly localized reaction would be without avail ? The 
same question arises in the case of the glycerin-fermenting bacteria 
to which Troili-Petersson 16 has ascribed an important rdle in the 
holing of Swedish cheeses. In fact experimental cheesemaking of the 
past, though not so thoroughly controlled as in the experiments of 
Troili-Petersson and those of Von Freudenreich and Jensen, bear 
evidence that any one of a number of gas-producing bacteria may 
provide the saturation, not to mention those reactions which Van 
Slyke and Hart 17 have proposed as contributing to the carbon dioxid 
in Cheddar cheese. On the other hand, any one of these may be the 
primary "inflator" and the other the secondary "saturator." 

In this connection it may be of interest to note a peculiar phe- 
nomenon met with in some experimental cheeses. A number of 
these made with "artificial" rennet by Mr. Doane were reported in 
their early stages to have begun a normal eye formation. Seldom, 
however, did this beginning develop into a normal holing. These 
cheeses were of small size, and, since it is known that small-sized 
cheeses for some reason not yet clearly defined seldom develop large 



SUMMAKY. 31 

eyes, the failure in these cases may on general principles be vaguely 
attributed to size. However that may be, it was found upon pump- 
ing out the dissolved gas that the amount was low. The three 
cheeses examined were 39-45, 39-11-2, and 46-4-1. (See Table 2, 
p. 12.) 

It is well known from the work of Jensen and others that the bac- 
teria found in " natural" rennet are often distinct from those found 
in "artificial" rennet. Since the cheeses under discussion were made 
with the latter, is it not possible that the reaction which started the 
eye formation was rendered inadequate because the gas-producing 
propionic bacteria, which might have saturated the cheese with 
carbon dioxid, were absent? That the observed holes were truly 
the beginnings of normal eyes, and were not a pinhole formation 
resulting from an initial gaseous fermentation of sugar, is evinced 
by the fact that hydrogen was absent. 

Exhaustive research alone can unravel this tangle; but it is hoped 
that the present investigation has provided both a clearer definition 
of the problem and a sound basis of fact. 

SUMMARY. 

1. The gases of normal "eyes" in Emmental cheese are exclu- 
sively carbon dioxid and nitrogen, and of these only the carbon 
dioxid is of significance. 

2. The nitrogen accompanying the carbon dioxid in normal eyes is 
that of air originally occluded in the curd at the time of manufacture. 

3. There sometimes occurs during the initial fermentation an evo- 
lution of gas characterized by the presence of hydrogen. This is 
believed to be due to the gaseous fermentation of sugar. 

4. The hydrogen from such an initial fermentation may sometimes 
linger to contaminate the gas of normal eyes. 

5. The two fermentations are distinct and are characterized by their 
gaseous products. The one is detrimental, the other that demanded 
of a good Emmental cheese. 

6. High oxygen-absorbing power combined with low permeability 
of the cheese to air render the interior thoroughly anaerobic, and conse- 
quently favorable to the growth of anaerobic bacteria. 

7. A comparison between the amount of carbon dioxid evolved 
and the total volatile fatty acids shows that the activity of the pro- 
pionic bacteria of Von Freudenreich and Jensen is not sufficient to 
account for all the carbon dioxid found. 

8. It was found that cheese is capable of retaining a very large 
amount of carbon dioxid. 

9. The possibility is suggested that there are two phases in the for- 
mation of normal eyes, a saturation of the body with carbon dioxid, 
and an inflation of eyes; and the bearing of this hypothesis on the 
production of gas by a specific cause is discussed. 



32 STUDY OF GASES OF EMMENTAL CHEESE. 

REFERENCES TO LITERATURE. 

1. [Bachler, C. Beitrage zur Erforschung des Gahrungsverlaufes in der Emmen- 

thaler Kasefabrikation. Schweizerisches Landwirtschaftlichen Centralblatt, 
Heft 1-6, 1896, cited by Jensen.] 

2. Boekhout, F. W. J., and Ott de Vries, Jan Jacob. Sur deux defauts du 

fromage d'Edam. Revue Generale du Lait, vol. 8, No. 14, p. 313-322; No. 
15, p. 347-356. Lierre, Sept. 30, 1910. 

3. Baumann, Fritz. Beitrage zur Erforschung der Kasereifung. Die Landwirt- 

schaftlichen Versuchs-Stationen, vol. 42, p. 181-214. Berlin, 1893. 

4. Becquerel, Paul. Sur la permeabilite aux gaz de l'atmosphere, du tegument 

de certaines graines dessechees. Academie des Sciences, Comptes Rendus, 
vol. 178, No. 22, p. 1347-1349. Paris, May 30, 1904. 

5. Dennis, L. M., and Hopkins, C. G. Die Bestimmung von Kohlenoxyd, Methan 

und Wasserstoff durch Verbrennung. Zeitschrift fur Anorganische Chemie, 
vol. 19, p. 179-193. Leipzig, 1898. 

6. Fischer, Emil, and Penzoldt, Franz. Ueber die Empfindlichkeit des Gerachs- 

sinnes. Annalen der Chemie, vol. 239, No. 1, p. 131-136. Leipzig, 1887. 

7. Fitz, A. Ueber Spaltpilzgahrungen. VI. Mittheilung. Berichte der Deut- 

schen Chemischen Gesellschaft. Vol. 13, p. 1309-1312. Berlin, 1880. 

8. Von Freudenreich, Edward, and Jensen, Orla. Recherches sur la fermenta- 

tion propionique dans le fromage d'Emmental. Annuaire Agricole de la Suisse, 
vol. 7, No. 4, p. 221-242. Bern, 1906. 

9. Gorini, Constantin. Sur la distribution des bacteries dans le fromage de Grana. 

Revue Generale du Lait, vol. 3, No. 13, p. 289-293. Lierre, Apr. 15, 1904. 

10. Graham, Thomas. Chemical and physical researches. Edinburgh, 1876. 

11. Jensen, Orla. Studien fiber die Lochbildung in den Emmenthaler Kasen 

Centralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten. 
Abteilung 2, vol. 4, No. 6, p. 217-222, Mar. 22; No. 7, p. 265-275, Apr. 1; No. 
8, p. 325-331, Apr. 26. Jena, 1898. 

12. Jensen, Orla. Studien uber die fluchtigen Fettsauren im Kase nebst Beit- 

ragen zur Biologie der Kasefermente. Centralblatt fur Bakteriologie, Para- 
sitenkunde und Infektionskrankheiten, Abteilung 2, vol. 13, No. 5/7, p. 161-170, 
Oct. 7; No. 9/11, p. 291-306, Oct. 21; No. 13/14, p. 428-439, Nov. 1; No. 16/17, 
p. 514-527, Nov. 11; No. 19/20, p. 604-615, Nov. 26; No. 22/23, p. 687-705, 
Dec. 10; No. 24, p. 753-765, Dec. 28. Jena, 1904. 

13. [Klenze. Handbuch fur Kasereitechnik, p. 198, cited by Jensen.] 

14. Marshall, Charles E. The aeration of milk. Michigan Agricultural Experi- 

ment Station. Special bulletin 16, Agricultural College, 1902. 

15. Nutting, P. G. The spectra of mixed gases. Astrophysical Journal, vol. 19, 

No. 2, pp. 105-110. Chicago, Mar. 1904. 

16. Troili-Petersson, Gerda. Experimentelle versuch liber die reifung und 

lochung des schwedischen giiterkases. Centralblatt fur Bakteriologie, Para- 
sitenkunde und Infektionskrankheiten, Abteilung 2, vol. 24, No. 13/15, p. 
343-360. Jena, Sept. 8, 1909. 

17. Van Slyke, Lucius Lincoln, and Hart, Edwin Bret. The relation of carbon 

dioxide to proteolysis in the ripening of Cheddar cheese. New York Agricul- 
tural Experiment Station (State) Bulletin 231. Geneva, 1903. 




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